Pulmonary epithelial cells are exposed to a diverse array of oxidants. Cytotoxicity occurs when the oxidant burden exceeds defenses but how injury induction occurs remains largely undefined. The aqueous fluid compartment (epithelial lining fluid; ELF) that covers airspace cells is initially contacted by inhaled oxidants. Endogenously derived oxidants (e.g. NO, ONOO- H2O2) are also secreted into or formed within it due to cellular activities and clinical therapeutics. An adaptive response, not exclusively linked to cellular alterations, occurs in a temporal and oxidant exposure-dependent manner. The oxidants NO2 and O3 undergo "reactive absorption" which couples uptake to their chemical reaction with similar ELF constituents. Despite this, recent data suggests NO2 & 03 display dissimilar ELF physiochemical interactions, making differential toxicity pathways probable. We hypothesize that: the mechanisms and extent of acute toxicity and development of adaptation are dependent on the specific chemical species that are formed within and diffuse through the ELF to contact the underlying epithelium. Thus, while heterogeneities in ELF chemistry likely govern the extent of injury from extracellular oxidants, the ELF contributions to the governance and mechanisms of acute injury and adaptation remain insufficiently defined. Due to their unique absorption but differential reaction/diffusion properties, we will employ NO2 & 03 as model ELF oxidants of disparate toxicity pathways. Our hypothesis will be tested by interrelated experimental aims utilizing novel investigational approaches that span from pure biochemical constructs to whole animal models. Accomplishment of the proposed aims will 1) delineate the temporal and spatial distribution of oxidative vs. nitrosative stress and their impact on acute cell injury from NO2 and 03, 2) characterize the ELF physicochemical conditions that alter dose/response relationships via modulating the formation and delivery of oxidative/nitrosative species to targets, and 3) define adaptation mechanisms relative to oxidative vs. nitrosative stress from extracellular oxidants and ascertain whether one form of stress imparts tolerance to the other. These proposed experiments will reveal new insights regarding the pathways of oxidant lung injury, especially related to the mechanisms that govern the magnitude of biological responses, and the potential to ameliorate lung injury